The use of diaminobenzidine for spectrophotometric and acrylamide gel detection of sulfite oxidase and its applicability to hydrogen peroxide-generating enzymes
DAB, in the presence of HRP, sulfite oxidase and FBS, polymerizes to a product with an absorption difference maximum at 352 nm. This reaction has been used as a sensitive and specific spectrophotometric assay for sulfite oxidase. Incubation of acrylamide gels containing sulfite oxidase with assay mixtures produces an insoluble product which precipitates where the enzyme is localized. This stain is at least as sensitive as the amido black protein stain and its specificity is such that no band is seen in the absence of substrate, and that only one band, migrating identically to purified enzyme is seen in rat liver homogenates. This polymerization reaction has been applied to other H2O2-generating enzymes and can be used to demonstrate the presence of these enzymes in the midst of other oxidases. DAB polymerization provides a sensitive spectrophotometric assay which can be used with either ultraviolet or visible optics. The application of this procedure to modified enzymes is discussed. © 1973.
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